Dr. Kevin Weeks requests four years of support to elucidate fundamental features of how RNA and protein molecules collaborate to perform RNA catalysis. The proposal focuses on a mode ribonucleoprotein catalyst formed by the bI5 group I intron, a catalytic RNA, and the CBP2 protein. Catalysis provides a sensitive proof of structure-function relationships in RNA-protein interactions. This system is simple enough to be understood in significant detail, yet it is sophisticated enough to illustrate principles fundamental to more complex RNA-protein machines like those responsible for the biologically and medically important processes of RNA biogenesis, polypeptide synthesis, and chromosome maintenance Specific Aims are as follows. (1) Obtain a detailed three dimensional view of the specific bI5 structures involved in interaction with CBP2. A novel method of covalent footprinting for mapping direct RNA-protein interactions in comple RNAs will be developed. RNA sequence and structural requirements for protein-facilitated catalysis will be examined using iterative in vitro selection. This work will also address compromises involved in requiring proteins to facilitate RNA catalysis. (2) Determine specific functional roles for the two structural domains that comprise the CBP2 splicing factor and assign functions to individual domains in the context of a specific, detailed, and comprehensive kinetic framework for protein-facilitated RNA splicing. The assignment of individual protein domains to specific activities will promote understanding of the general roles of splicing factors in RNA catalysis. (3) Establish a mechanistic framework for understanding how Suv3 protein functions to disassemble the CBP2-RNA complex. Facilitated recycling of CBP2 is essentia for additional rounds of splicing on a biologically relevant time scale. These studies will define principles governing reorganization of sophisticated RNA-protein complexes by proteins whose function is coupled to NTP hydrolysis.